Stem cells provide for the maintenance and regeneration of organs and tissues throughout life, and loss of the regenerative capacity of tissues such as blood and muscle has been attributed to decreased stem cell activity. The ability of stem cells to contribute to tissue homeostasis depends on the unique ability to generate both new stem cells (self-renewal) as well as specialized cell types (differentiation). The Aims of this proposal are to analyze changes in stem cell behavior during the aging process and to identify molecular mechanisms regulating these changes. We will also investigate the relationship between pathways regulating longevity and age-related changes to stem cells and the stem cell niche. Methods: We previously characterized the germ line stem cell (GSC) niche in the Drosophila testis, providing a system to study the intrinsic and extrinsic factors controlling stem cell behavior in vivo. Testes from males maintained with females will be analyzed for changes in stem cell behavior at 1, 30, and 50 days post-eclosion. Using readily available markers, we will analyze changes in gene expression and protein expression and localization in stem cells and niche cells using in situ analysis and immunofluorescence. GSC division will be assayed using BrdU incorporation as well as by marking cells through mitotic recombination. GSC behavior will be analyzed in wild type flies, flies in which the JAK-STAT pathway, which is necessary and sufficient for stem cell self-renewal, has been modulated, and in flies that have been characterized as being long-lived. Conclusions: In addition to providing insights into tissue homeostasis, studies focusing on age-related changes in stem cells and their specialized microenvironments, known as stem cell niches, will help to identify and overcome unique hurdles in the manipulation of tissue stem cells derived from older patients and to facilitate the development of techniques for regenerative medicine to treat aging-related diseases. Lay Summary: Evidence suggests that stem cell function decreases during the aging process, and the primary consequence is loss of tissue function. Tissue replacement therapies will rely heavily on expanding tissue stem cells in culture. This project seeks to characterize age-related changes in stem cells and the support cells that nurture them to facilitate the expansion and maintenance of stem cells in culture, an essential first step in the utilization of stem cells to treat aging-related diseases.